Renal Endothelial Cell‐Targeted Extracellular Vesicles Protect the Kidney from Ischemic Injury

Abstract Endothelial cell injury plays a critical part in ischemic acute kidney injury (AKI) and participates in the progression of AKI. Targeting renal endothelial cell therapy may ameliorate vascular injury and further improve the prognosis of ischemic AKI. Here, P‐selectin as a biomarker of ischemic AKI in endothelial cells is identified and P‐selectin binding peptide (PBP)‐engineered extracellular vesicles (PBP‐EVs) with imaging and therapeutic functions are developed. The results show that PBP‐EVs exhibit a selective targeting tendency to injured kidneys, while providing spatiotemporal information for the early diagnosis of AKI by quantifying the expression of P‐selectin in the kidneys by molecular imaging. Meanwhile, PBP‐EVs reveal superior nephroprotective functions in the promotion of renal repair and inhibition of fibrosis by alleviating inflammatory infiltration, improving reparative angiogenesis, and ameliorating maladaptive repair of the renal parenchyma. In conclusion, PBP‐EVs, as an ischemic AKI theranostic system that is designed in this study, provide a spatiotemporal diagnosis in the early stages of AKI to help guide personalized therapy and exhibit superior nephroprotective effects, offering proof‐of‐concept data to design EV‐based theranostic strategies to promote renal recovery and further improve long‐term outcomes following AKI.


Supplementary Fig. 11 | Targeting ability of PBP-EVs in vitro. a, Schematic diagram
showing study design of PBP-EVs targeting H/R injured HUVECs in vitro. b, Gluc imaging and quantification revealed H/R injured HUVECs internalized more PBP-EVs in vitro. n = 3.
Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison tests. c, Schematic diagram showed the study design of PBP-EVs penetration of H/R injured endothelium in vitro. d, LSCM images and quantification showed an increased internalization of PBP-EVs (Cy5.5, yellow) in HK2 (Alexa Fluor 488-labeled phalloidin, green, actin) in lower chambers. The nuclei were counterstained with DAPI (blue). Scale bar, 100 μm. n = 3.
Statistical analysis was performed using a two-tailed unpaired Student's t-test. All data are 12 expressed as mean ± s.d. * P < 0.05. BLI: bioluminescence imaging, LSCM: laser scanning confocal microscope.

Penetration of PBP-EVs through endothelial barriers
The penetration of EVs and PBP-EVs through a monolayer of endothelial cells was evaluated using a modified Transwell system (3472, Corning, Corning, NY) as previously described 2 .
HUVECs (1×10 4 cells per well) were seeded in the upper chambers of Transwell inserted into 24-well plates and cultured for an additional 7 days. The medium was replaced every other day. The confluence of the monolayer was checked to ensure that the next steps were conducted in the absence of gaps in cell junctions. Subsequently, HK2 (5×10 4 cells per well) were cultured on glass coverslips in the lower chambers (24-well plate) and the upper chambers with the HUVEC monolayer were inserted. After the Transwell systems were performed H/R administration, the Cy5.5/Gluc-labeled EVs or PBP-EVs (100 μg/mL) were added into the upper chambers and incubated for another 6 h. The media of the upper and lower chambers were collected and measured in the presence of CTZ by Gluc imaging.
HK2 cultured in the lower chambers were fixed and counterstained with Alexa Fluor 488-labeled phalloidin and DAPI for LSCM analysis.

Blocking effect of PBP-EVs
The adhesion of monocytes to injured endothelial cells was examined to assess the blocking effect of PBP-EVs on P-selectin. The normal or H/R administrated HUVECs (2×10 4 cells per well) were cultured on glass coverslips in 48-well plates and co-incubated with human monocyte THP-1 (5×10 3 cells per well). EVs or PBP-EVs (100 µg/mL) were added to one of the co-culture systems respectively and shaking incubated at 37 °C for 3 h. After removing 24 unbound THP-1 with PBS, the cells on glass coverslips were fixed with 2.5% glutaraldehyde and dehydrated with serial dilutions of ethanol. The morphology and amounts of THP-1 bound to HUVECs were observed and analyzed by scanning electron microscopy (SEM; Phenom, Shanghai, China).

Myeloperoxidase (MPO) assay of renal tissues.
The MPO activities of renal tissues were measured using the MPO assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). On day 3 post severe IRI, a quarter of the kidneys were homogenized to obtain 5% homogenate according to the manufacturer's instructions. The homogenates were centrifuged at 5,000 g for 5 min and the supernatants were removed for the MPO assay.

Endothelial cell function analysis
The tube formation assay and the wound healing assay were performed to analyze endothelial cell functions as previously reported 3 . Briefly, normal or H/R administrated HUVECs used for tube formation were seeded in growth factor-reduced Matrigel (Corning) coated 48-well plates and treated with EVs or PBP-EVs (100 μg/mL) for 6 h. The images of network structures were captured by a bright-field microscope (Olympus) and analyzed by ImageJ software to evaluate the proangiogenic capacity of HUVECs. Regarding the wound healing assay, a scratch wound was generated in the monolayer of normal or H/R administrated HUVECs and cultured for an additional 12 h with 100 μg/mL EVs or PBP-EVs. The images of wounds captured at 0 and 12 h were used to quantify the migration ratio using ImageJ